Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. A. AKT pathway mediates chemoresistance in NB Previously, we identified the PI-3 kinase pathway as mediating BDNF/TrkB-induced resistance to etoposide, vincristine and adriamycin. We evaluated the role of AKT in chemoresistance, because AKT is one of the primary downstream targets of PI-3 kinase and plays an important role in neuronal survival. Using genetic approaches, we determined that a dominant negative AKT could block the effects of BDNF activation of TrkB on NB chemoresistance. Moreover, this was the first report to demonstrate that activated AKT alone attenuates the cytotoxic effects of chemotherapy on NB cells. In collaboration with Dr. Dennis at the NCI, we tested a molecularly modeled phosphotidylinositol ether lipid analogue (PIA-6) that was rationally designed to target and inhibit the PH domain of AKT thus preventing its membrane localization and activataion. When PIA-6 was present, BDNF activation of AKT was diminished and chemosensitivity restored. This study demonstrated that inhibiton of AKT was sufficient to block the effects of activated TrkB. Moreover, even in the absence of TrkB activation, the AKT inhibitor more than doubled the efficacy of the cytotoxic agent utilized. Thus not only does this strategy target cells with activated TrkB, but it raises the possibility that any growth factor using Akt to mediate a survival signal may be effectively targeted with a PIA-6-like molecule. B. BDNF activation of TrkB via MAPK pathway decreases pro-apoptotic BH-3 protein BIM, which contributes to paclitaxel resistance. The ability of a tumor cell to survive an apoptoic insult or survive in a new microenvironment depends on both the external stress (chemotherapy, nutrient and O2 levels), the microenvironment (e.g. metastasize) and the cells internal signaling systems. Our study on Bim indicates that, depending on the chemotherapeutic drugs utilized, BDNF/TrkB activation attenuates the effects of cytotoxic agents by distinct signaling pathways. We used a candidate gene approach to investigate targets downstream of AKT involved in chemoresistance. Activated Akt phosphorylates and inhibits several pro-apoptotic proteins, such as Bad, caspase-9 and Forkhead transcription factors (FKHRL1, FKHR and AFX), leading to cell survival. We did not detect significant changes in phosphorylation or total protein levels for BAD or caspase 9 after BDNF activation of TrkB. In our AKT study, the genetic and pharmacologic inhibiton of AKT activity focused our attention on FKHRL1. FKHRL1 functions in apoptotic processes by regulating p27 interaction with caspase 8 and with the pro-apoptotic BH-3 protein BIM. As the NB cell lines under study did not express caspase 8, we focused on BIM. BIM is a target of AKT in many other systems. We found that BDNF/TrkB activation leads to a decrease in BIM levels that is mediated by activation of MAPK and not AKT. SiRNA mediated BIM knockdown had no effect on cisplatin/etoposide induced cytotoxicity. However, silencing of BIM or inhibition of the MAPK pathway sensitized NB cells to paclitaxel. Bim is thought to induce cell death by binding to LC8 cytoplasmic dynein light chain where it is sequestered with the microtubule-associated dynein motor complex in healthy cells. Certain apoptotic stimuli such as paclitaxel stabilize microtubules and disrupt the interaction between LC8 and the dynein motor complex, freeing Bim to translocate together with LC8 to Bcl-2 and neutralize its anti-apoptotic activity. In our study, reduction of Bim by Bim siRNA, decreases paclitaxel induced apoptosis. It is becoming increasingly apparent that different drugs or toxic insults utilize distinct apoptotic signaling paths just as survival signaling pathways use distinct pathways to attenuate death signals. Clinically, only 8/33 NB patients using an up-front phase II window approach had objective responses with paclitaxel which limited its potential utility when combined with current multi-agent regimens. Our study would indicate that drugs previously thought to have marginal activity may need to be re-evaluated in combination studies when appropriate signal transduction inhibitors become available. Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis A.HIF-1&#945; is a major target of Growth Factor regulation of VEGF in Neuroblastoma under normoxic conditions. The extent of angiogenesis and/or VEGF expression in neuroblastoma tumors correlates with metastases, N-myc amplification and poor clinical outcome. A number of studies indicate that targeting angiogenesis or VEGF expression has clinical utility. Metastasis is a multi-step process that is intimately linked with angiogenesis. Most of the mechanisms fundamental to metastasis, such as adhesion, proteolysis and migration are normal physiologic events that are utilized by neural crest cells during development and neurons during neuritogenesis. It is well established that hypoxia increases VEGF levels and over-expression of TrkA suppressed its expression in NB cells. The latter finding consistent with low levels of VEGF in TrkA-expressing, good prognosis NB tumors. Our interest in VEGF stemmed from observations in two preclinical studies we had performed in which decreases in VEGF expression and vascularity were noted in tumors of animals receiving either the histone deacetylase inhibitor, MS-27-275 or Gleevac. Since the molecular mechanisms regulating VEGF in NB cells had not been described, we decided to evaluate regulation of VEGF. Under normoxia, hydroxylation of HIF-1&#945; facilitates its interaction with VHL and subsequent degradation by the proteosome. Since mutations in VHL lead to constitutive expression of VEGF in some cancers, we sequenced the HIF-1&#945; coding exons in 15 NB cell lines. No mutations were in the coding exons were detected. We then determined that serum and serum-derived growth factors (such as PDGF, EGF and IGF-1) as well as BDNF stimulated VEGF expression in NB cells. Continuing our studies that first identified that the IGF/IGFR pathway regulated VEGF in NB cells under normoxia, we evaluated the role of the BDNF/TrkB pathway to regulate VEGF in NB. BDNF activation of TrkB-TK increases HIF-1&#945; expression via the PI-3Kinase and mTOR pathways. HIF-1&#945; is a major regulator of VEGF transcription. Using transient transfection analyses, we found that the BDNF/TrkB stimulated increases in HIF-1&#945; and VEGF in a manner depenent on HIF-1&#945; binding to HRE1 in the VEGF promoter-reporter. Silencing HIF-1&#945; expression with siRNAs targeted to HIF-1&#945; blocked BDNF-induced increases in VEGF promoter activity and decreased secreted VEGF levels. While hypoxia stimulates both HIF-1&#945; and HIF-2&#945;, BDNF stimulation did not alter the levels of HIF-2&#945; or HIF-1. Since we had shown in our study on IGF/IGFR that the the VEGF secreted by NB cells was capable of activating the Flk receptor on endothelial cells and stimulating their proliferation, we hypothesize that any growth factor whose tyrosine kinase receptor displayed a similar mechanism of activation would also stimulate HIF-1&#945; and VEGF levels